© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs223123. doi:10.1242/jcs.223123 SHORT REPORT Chk1-mediated Cdc25A degradation as a critical mechanism for normal cell cycle progression Hidemasa Goto1,*, Toyoaki Natsume2,3, Masato T. Kanemaki2,3, Aika Kaito4, Shujie Wang1, Esteban C. Gabazza5, Masaki Inagaki4 and Akira Mizoguchi1 ABSTRACT ATM is primarily activated by DNA double-strand breaks Chk1 (encoded by CHEK1 in mammals) is an evolutionarily conserved (DSBs). ATM phosphorylates and activates Chk2. Both kinases TP53 protein kinase that transduces checkpoint signals from ATR to Cdc25A phosphorylate p53 (encoded by ) resulting in p53 stabilization, during the DNA damage response (DDR). In mammals, Chk1 also which promotes p21 (also known as CDKN1A) expression. p21 controls cellular proliferation even in the absence of exogenous DNA binds and inhibits the cyclin and cyclin-dependent kinase (CDK) damage. However, little is known about how Chk1 regulates complexes leading to cell cycle arrest. By contrast, ATR is activated unperturbed cell cycle progression, and how this effect under by a broader spectrum of DNA-damaging stimuli, such as ultraviolet physiological conditions differs from its regulatory role in DDR. Here, (UV) radiation, DNA replication stress and inter-strand DNA we have established near-diploid HCT116 cell lines containing crosslinking reagents. ATR phosphorylates and activates Chk1, endogenous Chk1 protein tagged with a minimum auxin-inducible which in turn phosphorylates and inhibits Cdc25 phosphatases. degron (mAID) through CRISPR/Cas9-based gene editing. Cdc25 dephosphorylates inhibitory phosphorylation sites on CDK Establishment of these cells enabled us to induce specific and rapid (e.g. CDK1-Tyr15), resulting in CDK activation. Thus, Cdc25 depletion of the endogenous Chk1 protein, which resulted in aberrant inhibition ends up causing cell cycle arrest (Boutros et al., 2007). – accumulation of DNA damage factors that induced cell cycle arrest at S There are three Cdc25 paralogs (Cdc25A Cdc25C) in human cells. or G2 phase. Cdc25A was stabilized upon Chk1 depletion before the Cdc25A phosphorylation by Chk1 triggers its degradation in a accumulation of DNA damage factors. Simultaneous depletion of Chk1 ubiquitin/proteasome-dependent manner (Boutros et al., 2007; and Cdc25A partially suppressed the defects caused by Chk1 single Mailand et al., 2000). Phosphorylation of Cdc25B and Cdc25C by depletion. These results indicate that, similar to its function in DDR, Chk1 reduces their phosphatase activity (Boutros et al., 2007). Chk1 controls normal cell cycle progression mainly by inducing DDR signaling pathways were initially considered to play critical Cdc25A degradation. roles mainly in cells under exogenous DNA-damaging stress. However, previous studies showing that complete deficiency of KEY WORDS: Chk1, Cdc25A, Auxin-inducible degron, AID, Cell cycle ATR or Chk1 leads to aberrant accumulation of DNA damage with progression early embryonic lethality (Brown and Baltimore, 2000; Liu et al., 2000; Takai et al., 2000) challenged this general belief. INTRODUCTION Interestingly, knockout of Cdc25A also results in early embryonic DNA damage caused by exogenous and endogenous factors occurs lethality (Ray et al., 2007). In sharp contrast, mice with complete constantly in normal cells. DNA lesion activates a variety of cellular deficiency of Cdc25B or Cdc25C alone or with double knockout of DNA damage response (DDR) mechanisms, including the cell cycle Cdc25B and Cdc25C are viable without any relevant phenotype, checkpoints, DNA repair, apoptosis and senescence (Bartek and except for some meiotic abnormalities during oogenesis in Cdc25B- Lukas, 2007; Ciccia and Elledge, 2010; Jackson and Bartek, 2009). knockout mice (Chen et al., 2001; Ferguson et al., 2005; Lincoln The DDR requires the activation of two evolutionarily conserved et al., 2002). Cells derived from Cdc25B and Cdc25C double- protein kinase cascades, the ATM–Chk2 and ATR–Chk1 (Chk1 and knockout mice show no apparent abnormalities in their cell cycle Chk2 are encoded by CHEK1 and CHEK2, respectively, in profile or DDR (Ferguson et al., 2005). However, in human somatic mammals) pathways (Antoni et al., 2007; Awasthi et al., 2015; cells, Cdc25B reportedly plays critical roles in normal G2/M Blackford and Jackson, 2017; Flynn and Zou, 2011; Goto et al., transition and in the cell cycle checkpoint at the G2 phase, together 2015; Medema and Macu˚rek, 2012; Reinhardt and Yaffe, 2009; with Cdc25A (Cazales et al., 2005; Lammer et al., 1998; Timofeev Saldivar et al., 2017; Shiloh and Ziv, 2013; Zhang and Hunter, 2014). et al., 2010; Vázquez-Novelle et al., 2010). A previous study has shown that Chk1 inhibition using a Chk1 1Department of Neural Regeneration and Cell Communication, Mie University inhibitor or Chk1-specific siRNA induces accumulation of DNA Graduate School of Medicine, Tsu, Mie 514-8507, Japan. 2Department of damage and DDR even in the absence of exogenous DNA insults Chromosome Science, National Institute of Genetics, Research Organization (Beck et al., 2010; Syljuasen et al., 2005). However, whether the of Information and Systems (ROIS), Mishima, Shizuoka 411-8540, Japan. 3Department of Genetics, The Graduate University for Advanced Studies signaling pathway mediated by Chk1 in unperturbed cell cycle (SOKENDAI), Mishima, Shizuoka 411-8540, Japan. 4Department of Physiology, Mie progression and in DDR is similar remains unknown. To clarify University Graduate School of Medicine, Tsu, Mie 514-8507, Japan. 5Department of this, conditional gene-knockout- or RNA interference-mediated Immunology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan. knockdown of Chk1 could be used. However, it takes days to observe significant phenotypic changes using these technical *Author for correspondence ([email protected]) approaches, and thus it is extremely difficult to distinguish H.G., 0000-0002-6796-4467 whether the observed phenotype is due to Chk1 inhibition or to DDR. The use of Chk1 inhibitor is an alternative approach but, in Received 23 July 2018; Accepted 2 January 2019 this case, specificity of the inhibitor could become a problem. To Journal of Cell Science 1 SHORT REPORT Journal of Cell Science (2019) 132, jcs223123. doi:10.1242/jcs.223123 overcome these difficulties, in the present study, we established a We examined the effect of acute Chk1 depletion on cell human colon carcinoma HCT116 cell line carrying endogenous proliferation in the absence of exogenous DNA insult. As shown Chk1 that can be rapidly and specifically degraded in an indole-3- in Fig. 2A, each Chk1–mAID cell line showed only marginal acetic acid (IAA; a natural auxin)-dependent manner (Natsume changes in cell growth after 1 day in the presence of IAA compared et al., 2016; Nishimura et al., 2009). Rapid Chk1 depletion impeded to control DMSO. However, a 3-day culture in the presence of IAA cell proliferation and caused accumulation of aberrant DNA damage significantly reduced the cell proliferation of four independent marker proteins, such as γH2AX (H2AX phosphorylated at Ser139; Chk1–mAID cell lines but not that of their parental cells (Fig. 2A; H2AX is encoded by H2AFX). Cdc25A stabilization was observed also see Fig. 3C). FACS analyses revealed that a 3-day culture in the before the accumulation of DNA damage factors. In addition, the presence of IAA increases the sub-G1 fraction (a measure of the Chk1 depletion-associated phenotype was partially suppressed by apoptotic cell fraction) in Chk1mACl/mACl cell lines (Fig. 2B). depletion of both Chk1 and Cdc25A, suggesting that Chk1 plays a However, as judged by the Trypan Blue staining, the percentage of role in the normal cell cycle in the absence of exogenous DNA dead cells varied from less than 2% to more than 10% by experiment damage mainly by targeting Cdc25A. (data not shown; also see FACS data in Fig. 4A), suggesting that the increase in apoptosis may not be the main cause of growth defect by RESULTS AND DISCUSSION Chk1 depletion. FACS analyses also indicated that the G2/M The CRISPR/Cas9 gene editing technology enables insertion of a fraction was elevated upon IAA treatment for 3 days in DNA fragment (e.g. tags and drug-resistant markers) at a desired Chk1mACl/mACl cells but not in their parental cells (Fig. 2B). Since gene locus by a sequence-specific DSB induction and homologous only 1–2% mitotic cells were detected in these IAA-treated cells recombination repair (Cong et al., 2013; Mali et al., 2013; Ran et al., (Fig. S2A), Chk1 depletion induces cell cycle arrest at the G2/M 2013). We combined this gene knock-in technology to generate transition; we observed a similar tendency for the Chk1mAM/mAM human conditional cells using a donor harboring a minimum auxin- cell lines (data not shown). A 3-day culture in the presence of IAA inducible degron (mAID) tag (Natsume and Kanemaki, 2017; increased the level of p53 and p21 proteins but not the level of Natsume et al., 2016; Nishimura et al., 2009). As shown in Fig. 1A–C, CDK1 phosphorylated at Tyr15 (an inactive form of CDK1; we established a human near-diploid HCT116 colon carcinoma cell Fig. S2B) in Chk1–mAID cell lines. Therefore, Chk1 depletion line (Thompson and Compton, 2008) expressing an auxin-inducible results in p53 stabilization, which induces cell cycle arrest likely ubiquitin ligase component, Oryza sativa (Os)TIR1, and the through the induction of CDK inhibitor(s), such as p21. endogenous Chk1 protein fused with mAID and a monomeric We analyzed early changes after IAA treatment by Clover (mAID–mClover; mACl) (Natsume et al., 2016) or with immunoblotting (Fig. 2C; Fig. S3). The phosphorylation of mAID fused to five repeats of the Myc epitope tag (mAID–5Myc; H2AX-Ser139 (γH2AX) and Chk2-Thr68 gradually increased mAM) at its C-terminus. We established two independent clones per after IAA incubation; it peaked at ∼20–24 h in the observed range each genotype. Even in the absence of IAA, the protein level of (from 0 h to 24 h).